Plastic Surgery Research Council
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PSRC 60th Annual Meeting

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Differential Effects of Inflammatory Mediators TNFα, TGFβ1 on Cellular Differentiation in a Primary Murine Muscle Cell in vitro Model of Heterotopic Ossification
S. Alex Rottgers, M.D., Laurie B. Meszaros, PhD, Anand R. Kumar, M.D..
University of Pittsburgh, Pittsburgh, PA, USA.

Introduction: Heterotopic ossification (HO) is a pathologic condition of bone formation in extremity muscles. Systemic and local inflammatory conditions acting on muscle-derived progenitor cells (MDCs) may either support or alter myogenic differentiation and promote pathologic chondrogenic or osteogenic differentiation. The aim of this study is to evaluate the effects of inflammatory mediators (TNFα, TGFβ1) on MDC myogenic, chondrogenic, and osteogenic differentiation.
Methods: Primary mouse muscle cells were isolated from 8-week-old C57B/6J mice. Hindlimb muscles were sterilely processed and pre-plating on collagen-coated flasks for 2 hours to minimize fibroblasts. The non-adherent mixed population of MDCs was cultured in F-10 growth medium with no greater than 3-4 passages for amplification. Four populations of cells were analyzed via FACS for surface markers. 100,000 cells per well were cultured in DMEM-based proliferation medium (PM) alone or with 1 ng/ml TNFα or with 5 ng/ml of TGFβ1 alone or in various combinations.
Samples were collected after 3 days. RNA was isolated reverse transcribed to cDNA. Quantitative PCR analysis of MyoD, Sox9, and Osx, which are markers of myogenic, chondrogenic, osteogenic differentiation, was performed. Changes in target gene expression were expressed relative to untreated MDCs with expression normalized to GAPDH.
Results: FACS analysis revealed a mixed population of cells, with high Sca-1 and CD34 expression and low CD31, CD 56, CD144 and CD146 expression. After 3 days, all isolations of MDCs cultured with TNFα demonstrated unaffected expression of MyoD (1.02±0.50), Sox9 (0.56±0.30) and decreased expression of Osx (0.19±0.07). TGFβ1 cultured cells demonstrated decreased expression of MyoD (0.236±0.017), Sox9 (0.151±0.023) and Osx (0.001±0.002). Cells cultured with combined with TNFα/ TGFβ1 demonstrated decreased expression of MyoD (0.158±0.036), Sox9 (0.131±0.020) and Osx (0.127±0.027).
Discussion: We have demonstrated that systemic inflammatory mediators can significantly affect MDCs transcription of myogenic, chondrogenic, and osteogenic differentiation factors. TNFα may protect against pathologic differentiation as it did not significantly affect in vitro myogenic or chondrogenic differentiation but suppressed osteogenic differentiation. Cells cultured in TGFβ1 alone demonstrated decreased in vitro myogenic, chondrogenic, and osteogenic differentiation. Cells cultured with both TNFα/ TGFβ1 demonstrated similar in vitro myogenic, chondrogenic, and osteogenic suppression as cells treated with TGFβ1 alone.


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